Methods and apparatus for optical analysis of samples in biological sample containers

ABSTRACT

An apparatus and method for optically analyzing samples in a biological sample container containing samples arranged at different locations on the base of the container. An optical acquisition device is provided comprising a detector and an objective. The position of the upper and lower surfaces of the base at each of the sample locations is determined by a confocal polychromatic displacement sensor. Light is collected from each of the sample locations by adjusting the focal plane to be coincident with, or vertically offset from, the upper surface of the base, as determined from the displacement sensor. This allows for rapid scanning of large numbers of samples in a multi-well plate or other biological sample containers.

BACKGROUND OF THE INVENTION

The present invention relates to automated methods and apparatus foroptically analyzing samples in biological sample containers such as wellplates.

Biological samples such as animal cells, in particular mammalian cells,are commonly cultured in biological sample containers such as wellplates (sometimes called microtiter plates or microplates), omni trays,Q-trays and Petri dishes. Much of the processing of the samples can beperformed automatically using robotic apparatus that can delivercontainers to and from various stations at which the samples can beobserved and imaged using camera equipment, and transferred to othercontainers using an array of pins on a movable mechanical head.

FIGS. 1A and 1B schematically illustrate an example biological samplecontainer 1 in the form of a well plate with a plurality of wells 5 in a4×3 array. More typically a well plate used for automated processes on arobotic platform will have an array of wells, e.g. 6, 24, 96, 384, 1536,3072 or 6144 etc wells, but may sometimes only have a single well. Thespacing between the wells and/or the external dimensions of the platesrelevant for handling, typically conform to the standard of the Societyfor Biomolecular Screening (SBS) adopted by the American NationalStandards Institute (ANSI) or derivates and extensions thereof used inthe industry. The ANSI microplate standards include: SBS-1 2004:Footprint Dimensions; SBS-2 2004: Height Dimensions; SBS-3 2004: BottomOutside Flange Dimensions; and SBS-4 2004: Well Positions of 96, 384,and 1536 well plates. The contents of these microplate standards areincorporated herein by reference, in particular the relevant dimensions.

For example, according to the SBS-4 standard, a 96 well plate has wellsspaced apart in a square grid by 9 mm. The corresponding dimensions for384 and 1536 well plates according to the SBS-4 standard are 4.5 mm and2.25 mm respectively. Other well plates can have their well spacingdimension calculated on this basis, even if not explicitly covered bythe SBS-4 standard, e.g. 24 well plates can be provided with an 18 mminter-well spacing as an extrapolation of the ANSI standard.

Further, according to the SBS-1 standard, a well plate should haveexternal dimensions of 127.76 mm (length)×85.48 mm (width)±certainspecified tolerances.

References to standard dimensions in relation to well plates made inthis document are thus made with reference to the above true standardsdefined by ANSI, and also derivates from and extensions of thesestandards used in the industry, as well as covering new standards thatmay be defined for well plates in the future.

Referring to FIG. 1A, the biological sample container 1 is a tray-likecontainer having a top surface 2 with a number of wells 5 foraccommodating biological or chemical samples. FIG. 1B is a schematiccross section along the line A-A′ of FIG. 1A. It can be seen from FIG.1B that each of the wells 5 comprises a side wall 3 and a base 4. In thepresent case, the well plate 1 is one suitable for optical imaging sothe base 4 of each well 5 is transparent, and optionally the side wall 3also.

For successful imaging, it is necessary to be able to accuratelyposition the sample in the field of view of an imaging camera, and tofocus the camera on the plane of interest. For well plate imaging inautomated processes, the focusing of the imaging camera needs to becarried out in an automated way, and autofocus systems are generallyused in the art for this purpose.

U.S. Pat. No. 6,130,745 [1] and U.S. Pat. No. 6,441,894 [2] describe aprior art technique for focusing a laser beam used to excitefluorescence in cell colonies cultured in wells in a well plate. It isimportant to accurately position a tightly focused beam within the cellcolony so as to avoid exciting fluorescence in unbound fluorescentmarkers outside the colony. The method involves focusing the laser beamnear the lower surface of the base of a well, and detecting lightreflected back. The basic principles of this method are schematicallyillustrated in FIGS. 2A to 2C. A well 15 contains a solution 18 whichmay contain sample cells to be imaged (not shown). FIG. 2A illustratesthe arrangement at a first time t=t₁, FIG. 2B illustrates thearrangement at a second time t=t₂, and FIG. 2B illustrates thearrangement at a third time t=t₃. In each case, the well 15 comprises aside wall 13 and a base 14. The base has a finite thickness as definedby an upper surface and a lower surface thereof. Referring first to FIG.2A, a laser is disposed beneath the well 15, and light emitted from thelaser is focused by a lens 16 to a focal point 17 near the lower surfaceof the base 14 of the well 15. Then, from the time t₁ to the time t₃,the focal point 17 is scanned upwards. The reflected light intensityreaches a maximum when the light is focused on the lower surface. Thisoccurs at around the time t₂ as shown in FIG. 2B. Thus, the lowersurface of the well base is detected. The focus is then advanced upwardsby at least the known thickness of the base so that the sample volumedefined by the well is focused. It is noted that the base thickness isknown from the specification of the well plate provided by the wellplate manufacturer. It is further noted that imaging of well plates frombelow, as shown in this prior art system, is generally preferred for anumber of reasons. First, there is generally better optical access frombelow, since the side walls do not need to be avoided. Second, it avoidshaving to image through the solution contained in the well. This isproblematic, since the volume of liquid solution varies and thus theheight of the upper surface of the liquid. Moreover, the upper surfaceof the liquid can move and inherently is not flat owing to meniscuseffects.

With regard to focusing a camera to image the cells, a standardautofocus system may be adequate. However, for a container requiringmany images, such as a well plate comprising 96, 384 or 1536 wells, itcan be very time-consuming to refocus the camera for each well. This isparticularly problematic if no stains or fluorescent tags are used tohighlight the cells; the visual contrast between the cells and theirsurroundings can be insufficient for the optical feedback in theautofocus system to function efficiently. As an example, under theseconditions it can take over an hour to image each well in a 96-wellplate by refocusing the camera for every well, which is inconvenientlyslow for an automated system intended to handle many cell samples.Examples of such systems include the Nikon PFS (“perfect focus” TM)system and the Olympus ZDS (“zero drift” TM) system.

FIG. 3 schematically illustrates another prior art laser-based autofocussystem of the type which is used by Molecular Devices Corporation intheir automated microscope system sold under the trade name ImageXpressMICRO (TM). A laser beam 27 from a laser 26 is directed at a glancingangle towards the transparent base 24 of a sample container 21, and thereflections from both an upper surface 24 a and a lower surface 24 b ofthe base 24 are detected by a detector 28. As a result of the laserbeing directed at the base at a glancing angle, the reflected light fromthe upper surface 24 a will take a path 29 b which is parallel to, butoffset from, a path 29 a taken by the reflected light from the lowersurface 24 b. The position of each path can be used to provide anindication of the location of the respective surface, and the distancebetween the two paths can be used to determine the thickness of thebase. While this technique may function adequately for a perfectly flatbase having perfectly flat upper and lower surfaces, in reality this maynot be the case. For example, the biological sample container may bebowed, which will cause the light beams 29 a and 29 b to be divergentand no longer allow accurate measurement of the base thickness.

FIGS. 4A and 4B schematically illustrate another prior art solutionwhich is described in US20070009395A1 [3]. In this solution, a wellplate is held in a specially designed vacuum bed and sucked down so itsbase is pressed against an optically flat surface, thus ensuring thatall wells lie in the same plane and thereby obviating the need to focuson every well prior to imaging. Referring to FIG. 4A, the holderincludes a vacuum bed having an optically flat planar surface 36 forreceiving a lower surface 34 of a biological sample container 31 and aperimeter seal 32 surrounding the vacuum bed. The seal is dimensioned toreceive the lower perimeter edge of a standard well plate 31. The holderincludes an exhaust outlet for evacuating the space under the well plate31 so that the well bases 34 are urged against the optical flat 36,thereby ensuring that the bases of all the wells are coplanar with eachother as shown in FIG. 4B. This enables many or all of the samples in acontainer to be imaged sequentially without the need to refocus animaging camera for every sample. Instead, the camera can be focused justonce on one sample in one region of the container, and the focusretained for imaging the remainder of the container. This significantlyspeeds up the time needed for handling each container. However, thismethod is only as accurate as the manufacturing tolerances in thethickness of the material between the base of the well plate and thebase of each well.

SUMMARY OF THE INVENTION

The invention provides a method of optically analyzing samples in abiological sample container, comprising the steps of:

providing a biological sample container containing a plurality ofsamples located at respective sample locations distributed over thebiological sample container, each sample location being coincident with,or vertically offset from, a base of the biological sample container,wherein the base is defined by upper and lower surfaces;

providing an optical acquisition device comprising a detector and anobjective which collectively define a focal plane for opticalacquisition;

measuring the position of at least one of the upper and lower surface ofthe base at each of the sample locations by focusing a continuum ofwavelengths of polychromatic light to a continuum of respectivepre-calibrated positions along an axis extending through the base, andby collecting, preferably confocally, and spectrally decomposing thosecomponents of the polychromatic light scattered from said axis; and

collecting light from each of the sample locations by adjusting thefocal plane to be coincident with, or vertically offset from, the uppersurface of the base based on the position of the at least one of theupper and lower surface of the base measured at that sample location.

The invention further provides an apparatus for optically analyzingsamples in a biological sample container, the biological samplecontainer containing a plurality of samples located at respective samplelocations distributed over the biological sample container, each samplelocation being coincident with, or vertically offset from, a base of thebiological sample container, wherein the base is defined by upper andlower surfaces, the apparatus comprising:

a container station in which a biological sample container can bearranged;

an optical acquisition device comprising a detector and an objectivearranged to view a biological sample container arranged in the containerstation from below, the positions of the detector and objectivecollectively defining a focal plane for optical acquisition;

a focal plane sensor arranged to view a biological sample containerarranged in the container station from below and comprising: (i) apolychromatic light source operable to generate polychromatic light overa range of wavelengths; (ii) a focusing arrangement with defined axialchromatism arranged to focus respective wavelengths of the polychromaticlight to respective pre-calibrated positions along an axis that extendsthrough where the base of a biological sample container arranged in thecontainer station would be; (iii) a detection unit comprising aspectrometer arranged to spectrally isolate components of saidpolychromatic light scattered from said optical axis, preferably withthe aid of a confocal collection aperture; and (iv) a data processingunit operable to determine the position of at least one of the upper andlower surface of the base from the isolated components of the scatteredpolychromatic light output by the spectrometer;

a positioning apparatus operable to adjust the focal plane of theoptical acquisition device relative to the container station; and

a controller operable to control the focal plane sensor, opticalacquisition device and positioning apparatus to: (i) determine a desiredfocal plane for each sample location with reference to the determinedposition of the at least one of the upper and lower surface of the baseat that sample location; and (ii) use the optical acquisition device tocollect light from each of the sample locations with the focal planeadjusted to its desired setting.

The predetermined relationship between wavelength and position on theoptical axis can be known in advance very accurately by factorycalibration, and thus the position of an intersection between the baseof the sample container and the optical axis can also be known veryaccurately by detecting the wavelength of reflected light. In this way,the measured position of the base on the optical axis can be used as areference for accurately determining a focal plane for imaging sampleswithin the sample container. This method of determining the position ofthe base of a sample container is particularly suitable because it canbe achieved very rapidly compared with conventional autofocus andfocused laser methods, in which it is necessary to effectively searchfor the base of the sample container by varying the focal length of theautofocus mechanism over time until the base of the sample container hasbeen detected. In particular, the present technique is able tosimultaneously search all positions along the optical axis by focusinglight of different wavelengths along the optical axis and detecting thewavelength of light reflected back from the intersection between thebase and the optical axis.

The present technique has been specifically developed for well plates inwhich case the sample locations are defined, or at least confined to,known positions of wells in a standard well plate. However, otherbiological sample containers may be used such as omni trays, Q-trays,Petri dishes and the like, and may be useful for imaging individualcells or colonies of cells distributed over a Petri dish or othercontainer type, the coordinates of which have been ascertained bystandard imaging, as known from imaging devices used in colony pickers.

The detector may be an imaging camera for obtaining an image of a samplewithin said biological sample container through said base.

The objective is preferably a lens, such as a single lens, but may be amirror arrangement, or a lens-mirror combination.

In the apparatus, the controller is operable to determine a desiredfocal plane for each of a plurality of the sample locations prior tocollecting light using the optical acquisition device from those samplelocations. The plurality of sample locations could be all the samplelocations, or a subset such as one or more rows or columns of wells of awell plate, or a contiguous area of wells.

The controller may instead or also be operable to determine a desiredfocal plane for a sample location and collect light from that locationusing the optical acquisition device prior to moving to the next samplelocation. This might be the preferred alternative in a Petri dish orother container when detecting cell colonies distributed over the dish.

Generally, both the upper and lower surfaces of the base will bedetected. However, for some sample positions the upper surface positionmay not be accurately obtained, since this is generally more difficultto measure than the lower surface position. To take account of thispossibility, the controller is preferably operable to take the focalplane for each sample location with reference to the measured uppersurface position, if available, and otherwise with reference to themeasured lower surface position. An alternative solution to the sameproblem is for the controller to be operable to take the focal plane foreach sample location with reference to a support surface on which thebase is in contact offset by a base thickness value computed from thedifference between the measured upper and lower surface positions, ifavailable, and otherwise with reference to the support surface. Forexample, an accurate measurement of the lower surface position can beobtained by flattening the base of the biological sample containeragainst an optical flat according to the disclosure of US20070009395A1[3].

The detector may be an array detector for imaging the sample location,for example a CCD sensor or multi-channel plate sensor. Alternatively,the detector may not have position resolution and be used, for example,for collecting non-spatially resolved fluorescence from the samplelocation.

The container station may be adapted to accommodate standard well plateshaving standard external dimensions, or other standard biological samplecontainers.

The controller may be operable to control the focal plane sensor,optical acquisition device and positioning apparatus assuming that thebiological sample container is a well plate having a standard number ofwells distributed in standard positions over the well plate.

The controller may be operable to control the focal plane sensor,optical acquisition device and positioning apparatus assuming that thesample locations are positions of single cells or cell colonies providedby a cell or cell colony imager.

In the context of a well plate, the lower surface corresponds to theexternal surface of the bottom of a well, whereas the upper surfacecorresponds to the inner surface of a well. If the intersection betweenthe base of the biological sample container and the optical axis is atthe inner surface of the biological sample container, the focal planeposition can be set to the inner surface, or to a position within thesample container just above the inner surface of the base, which can beachieved by adding a predetermined offset value to the determinedposition on the optical axis.

Alternatively, if the intersection between the base of the biologicalsample container and the optical axis is at the outer surface of thebiological sample container, a predetermined offset distance to be addedto the position of the outer surface. In this case, a position for thefocal plane is selected in dependence on a thickness of the base of thebiological sample container.

The thickness of the base of the biological sample container, that isthe thickness between the bottom of a well and the bottom of thecontainer itself, may be determined by receiving light reflected from anintersection between the outer surface and the optical axis, and lightreflected from an intersection between an inner surface of the base ofthe biological sample container and the optical axis, determining aposition on the optical axis of each of the outer surface and innersurface of the base in dependence on a wavelength of light received fromeach intersection, and determining a thickness for the base from adifference in position on the optical axis between the upper surface andlower surface of the base. The thickness measurements may be filtered toremove spurious measurements, and averaged to produce a mean thicknessvalue for the base of the container. Alternatively, the thickness of thebase may be known in advance from the manufacturer.

Two main modes can be used for conducting the focusing and imagingsequence. In a first mode, the controller aligns the focal plane sensorand the biological sample container so that the focal plane sensor is ata position for imaging a region of the biological sample container, thenoperates the focal plane sensor to determine a position of a focal planefor imaging at the region of the biological sample container, thenaligns the imaging camera and the biological sample container so thatthe imaging camera is at the position for imaging the region of thebiological sample container, and then operates the imaging camera toimage a sample within the region of the biological sample container atthe focal plane determined by the focal plane sensor.

The controller can image a plurality of different regions of thebiological sample container by repeatedly performing the sequence ofaligning the focal plane sensor, operating the focal plane sensor,aligning the imaging camera and operating the imaging camera.

Alternatively, the controller may align the focal plane sensor and thebiological sample container so that the focal plane sensor is at aposition for imaging a region of the biological sample container, andoperate the focal plane sensor to determine a focal plane for imaging atthe region of the biological sample container. In this case, thecontroller repeatedly aligns and operates the focal plane sensor withrespect to the biological sample container to image a plurality ofdifferent regions of the biological sample container, generates a focalplane profile for the plurality of different regions, and operates theimaging camera to image samples within the plurality of differentregions of the biological sample container at respective focal planesdetermined in accordance with the focal plane profile.

According to another aspect of the invention, there is provided a methodof acquiring images of samples in a biological sample container,comprising the steps of:

providing a biological sample container having a base which is at leastpartially optically transparent;

determining a focal length for imaging a sample within said biologicalsample container through said optically transparent base;

focusing at said determined focal length; and

obtaining an image of said sample;

wherein said focusing step comprises

focusing a continuum of different wavelengths of light from apolychromatic light source to a continuum of different positions on afocal axis, a relationship between said wavelengths and said positionsbeing predetermined;

receiving light reflected from an intersection between said base of saidbiological sample container and said focal axis;

detecting a wavelength of said received light;

determining a position on said focal axis corresponding to said detectedwavelength in accordance with said predetermined relationship betweenwavelength and position; and

determining said focal plane position in dependence on said determinedposition on said focal axis.

The holder is generally applicable to biological sample containers, suchas omni-trays, Q-trays and Petri dishes. However, it is particularlyadvantageous where the biological sample container is a well plate,which can hold a great number of samples all of which need to beprocessed, preferably in an automated manner.

The samples may be cells, in particular animal cells. Moreover, thecells could be individual cells, colonies of cells, cell monolayers orother kinds of cell aggregates. The method can be used for pickingvaluable or interesting cells or colonies of cells from a cellpopulation. The cells may be 1 to 50 in number in the case of individualcells, or much greater in number in the case of colonies.

The samples may be cells, in particular animal cells. Moreover, thecells could be individual cells, colonies of cells, cell monolayers orother kinds of cell aggregates.

Various other aspects and features of the invention are defined in theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention and to show how the same maybe carried into effect reference is now made by way of example to theaccompanying drawings in which:

FIGS. 1A and 1B schematically illustrate a standard well plate;

FIGS. 2A to 2C schematically illustrate a prior art laser based focusingmethod for imaging samples in a biological sample container such as awell plate;

FIG. 3 schematically illustrates another prior art laser based focusingmethod for imaging samples in a biological sample container such as awell plate;

FIGS. 4A and 4B schematically illustrate a further prior art method ofaligning samples in a well plate using a vacuum bed with an opticalflat;

FIG. 5 schematically illustrates an apparatus for handling andprocessing biological samples in biological sample containers;

FIG. 6 schematically illustrates an imaging apparatus according to anembodiment of the invention;

FIG. 7 schematically illustrates a displacement sensor according to anembodiment of the invention;

FIG. 8 schematically illustrates the use of a chromatic confocaldisplacement sensor for detecting the position of a base of a biologicalsample container according to an embodiment of the invention;

FIG. 9 schematically illustrates a first scanning sequence according toan embodiment of the invention;

FIG. 10 schematically illustrates a second scanning sequence accordingto an embodiment of the invention;

FIG. 11 is a schematic flow diagram which illustrates the first scanningsequence in an embodiment in which the top surface is detected and usedto set the focal plane of a well in a well plate;

FIG. 12 is a schematic flow diagram which illustrates the first scanningsequence in an embodiment where the bottom surface is detected and usedin combination with derived thickness information to set the focal planeof a well in a well plate;

FIG. 13 is a schematic flow diagram which illustrates the secondscanning sequence in an embodiment where the top surface is detected andused to set the focal plane of a well in a well plate; and

FIG. 14 is a schematic flow diagram which illustrates the secondscanning sequence in an embodiment where the bottom surface is detectedand used in combination with known thickness information to set thefocal plane of a well in a well plate.

DETAILED DESCRIPTION

FIG. 5 is a perspective view of an apparatus for handling and processingbiological samples that embodies aspects of the present invention.However, it is to be understood that the various aspects of theinvention may be used with alternative apparatus, containing fewer ormore features for handling and processing samples and/or for handlingsamples in alternative biological sample containers.

The apparatus 110 may be understood as a robot for cell picking havingan integrated imaging camera. The apparatus can be subdivided notionallyinto two-half spaces, one above and one below a main bed 112 which issupported by a frame 114. The main bed 112 is mounted on linearpositioners (not shown) so as to be movable relative to the frame 114 inthe x and y directions, under the control of a controller (not shown).The controller may be a computer connected by electronic links usingstandard interface protocols to various automated components of theapparatus 110, with control of the apparatus effected by controlsoftware resident in the computer.

A cell picking head 118 is provided that comprises a plurality of hollowpins for aspirating animal cells such as mammalian cells, allowing cellsto be picked from one container and deposited in another container. Thecell picking head 118 is suspended over the main bed 112 from a gantry120 by way of a head positioning system made up of x-, y- and z-linearpositioners operable to move the cell picking head 118 over the main bed112. The gantry 120 is mounted on a rail 122 attached to the frame 114and can slide therealong to give further movement of the cell pickinghead 118 relative to the main bed 112. All movements can be controlledby the controller.

The main bed 112 contains a plurality of stations 116 (in this caseeight) being apertures adapted to receive biological sample containers(not shown) and possibly also components such as a wash/dry station forcleaning the pins after picking. In this example, the apertures arerectangular and shaped to received biological sample containers in theform of well plates, such as plates containing 96, 384 or 1536 wells.However, other containers such as omni trays, Q-trays and Petri dishesmay also be handled by providing suitable stations, or using adaptersthat fit into the well plate stations to hold the containers. The x andy movement of the main bed 112 can be used in conjunction with themovement of the cell picking head 118 to accurately position the pins ofthe head 118 over particular wells in the well plates. Also, the mainbed 112 can be moved to the right hand end of the frame 114 (asillustrated) to bring a container imaging station 130 to an imagingassembly 124 that includes an optical acquisition device.

The imaging assembly 124 is mounted on the frame 114, and comprises alight beam source 126 positioned in the upper half space to direct lightdownwards onto a well plate held in the container imaging station 130.The optical acquisition part includes a detector in the form of animaging camera 128 positioned in the lower half space and directedupwards to image cells cultured in the well plate when illuminated bythe light source 126, the imaging being through suitable focusingoptics, namely an objective (not shown) which may be a single lens ormultiple lens combination. Equivalent mirror components could also beused. The imaging assembly also includes a focal plane sensor 129mounted next to the imaging camera 128. The imaging station 130 includesa holder 132 mounted on the main bed 112 for holding a biological samplecontainer, in this case a well plate.

With the source above the sample and the detector below, a transmissionmode optical system is formed. It will be appreciated that a reflectionmode optical system may also be used in which the source and detectorare both below the sample, or both above. For example, the source anddetector optical paths may be combined by a semi-silvered mirror, beamsplitter or other known optical components. Reflection mode systems aredescribed in US2006164644A1 [5] and US2006166305A1 [6], the contents ofboth of which are incorporated herein by reference in their entirety andin particular in respect of the disclosed reflection mode opticalconfigurations on a robotic platform.

FIG. 6 shows a perspective view of the imaging assembly 124 of FIG. 1.The imaging assembly comprises a light beam source 126 mounted at theupper end of a bracket 100 and positioned to direct a beam ofilluminating light downwards to an imaging camera 128 mounted at thelower end of the bracket 100. The bracket 100 is configured for mountingof the imaging assembly on the frame 114 of the apparatus 110 so thatthe beam source 126 is above the main bed 112 and the camera 128 isbelow the main bed 112 (see FIG. 5). In this way, a biological sample ina well of a well plate 150 held in the imaging station 130 of the mainbed 112 can be positioned in the beam path for imaging using the camera.In addition to the camera 128, a focal plane sensor 129 is providedadjacent to the camera which serves to determine a suitable focal planefor the camera 128 to use to image samples in the biological samplecontainer 150.

The light beam source 26 may be an LED (Light Emitting Diode) or one ofa variety of other light sources may be used including conventionalfilament light sources, superfluorescent LEDs, diode lasers, other typesof solid state laser or gas lasers. Fixed wavelength or tunable diodelasers may be used. The light source 126 emits light downwards toprovide a beam incident on a converging lens 127 supported under thelight source. The camera 128 is directed upwards to image samples in awell plate 150 held in the light beam by the main bed 112. The camera128 includes a DC motor 142 operable to control the focus of the camerain response to focal plane information output from the focal planesensor 129. The focal plane sensor 129 and the camera 128 are mountedtogether on the bracket 100 at a known separation. In an alternativeembodiment, a focal plane sensor could be provided on a separate bracketand be moveable with respect to the camera.

A controller, which may be a combined controller operable to control allfeatures of the apparatus 110, or a dedicated imaging controller, isconnected to the imaging assembly 124. The controller will send thenecessary instructions to the various parts of the imaging assembly 124for obtaining images of samples. Namely, a well of a well plate (or asample-containing region of a different container) is aligned with theoptical axis of the focal plane sensor 129 to determine a focal planefor imaging, and then into the field of view of the camera and intoalignment with the beam source 126, using the x and y movement of themain bed 130.

The light source is arranged so that the base of the well is illuminatedwith a light beam formed by the lens 127, and the camera 128 takes animage of the illuminated well base at the focal plane determined by thefocal plane sensor 129. The light source 126 may be switched on and offto provide separate illumination for each image, or may be left oncontinuously, since the opening of the camera shutter will determine theexposure. In the former case, there is no need to synchronize theillumination with the camera operation. Instead, the camera shutter canbe opened for an exposure time that is long compared to a much shorterillumination time, timed to occur during the camera exposure time.Alternatively, the camera shutter can be left open for the duration ofthe imaging process, and the light source switched on for a briefexposure for each of the well plate positions.

Although the imaging has been described in terms of moving the wellplate, it is to be understood that the required relative movements mayalso be achieved by keeping the well plate stationary and moving thelight source and lenses and the camera instead, or by combiningmovements of these components with movements of the well plate.

FIG. 7 schematically illustrates a displacement sensor which can formpart of a focal plane sensor. The displacement sensor uses a chromaticconfocal optical configuration to detect the relative position of thebase of a biological sample container. The relative position of the baseof the biological sample container can be used to control an imagingcamera to focus at the detected position of the base (or at a selectedposition relative to the detected position of the base) because theposition of the displacement sensor with respect to the imaging camera(and in particular its objective lens) is known. The displacement sensorincludes a light source 310 which emits polychromatic light, that is,light having a continuous spread of wavelengths, for example whitelight. The displacement sensor also includes a lens 320 which focusesthe light emitted from the light source 310 at different foci along anoptical axis 330 which bisects the lens 320. The lens 320 intentionallyexhibits substantial axial chromatism which causes different wavelengthsof light to be refracted through the lens by different amounts. In otherwords, the lens 320 has a refractive index which varies as a function ofwavelength. As a result, light emitted from the light source 310 isfocused by the lens 320 to provide a continuum of monochromatic focalpoints distributed along the optical axis 330. In particular, five focalpoints d₁, d₂, d₃, d₄ and d₅ are shown in FIG. 7, these being atwavelength λ₁, wavelength λ₂, wavelength λ₃ wavelength λ₄ and wavelengthλ₅ respectively. Each of these wavelengths is focused to a knownposition on the optical axis 330, and in particular to a known distancefrom the lens 320. Specifically, the wavelength λ₁ corresponds to adistance d₁ from the lens 320, the wavelength λ₂ corresponds to adistance d₂ from the lens 320, the wavelength λ₃ corresponds to adistance d₃ from the lens 320, the wavelength λ₄ corresponds to adistance d₄ from the lens 320, and the wavelength λ₅ corresponds to adistance d₅ from the lens 320. It will however be appreciated that thefive focal points identified above are not the only points at whichlight is focused, but rather are example focal points of a continuum offocal points generated along the optical axis 330.

When an object intersects the optical axis 330 at one of the focalpoints, light striking the object may be scattered back through the lens320 back towards the light source 310, where it is reflected by ahalf-silvered mirror 340 towards a detection unit comprising a confocalaperture 350, spectrometer 360 and processor 370. Other forms of beamsplitter may be used, as will be understood in the art. Equivalent fiberoptic splitter components could also be used.

The light scattered back towards the spectrometer 360 is filteredthrough the confocal aperture 350 which acts as a spatial filter. Thespectrometer 360 detects the intensity and wavelength of the light whichit receives, and performs a spectral decomposition of the lightscattered from the optical axis to identify the prominent wavelengths.The prominent wavelengths may be those wavelengths at which theintensity of received light exceeds a predetermined threshold. More thanone prominent wavelength may occur if an object intersecting the opticalaxis is at least partially transparent, because such an object wouldprovide at least two relevant intersections, these being the entry andexit points of the optical axis 330 through the object. This propertycan be used to detect not only the distance of the object from the lens320, but also the thickness of the object. The prominent wavelengths arethen passed to a processor 370 in which is stored a predeterminedcorrespondence between distance relative to the lens 320, or other fixedpoint along the optical axis, and wavelength. The distances whichcorrespond to each of the prominent wavelengths identified by thespectrometer 360 are then determined. In the context of the arrangementof FIGS. 5 and 6, where the object intersecting the optical axis 330 isthe base of a biological sample container, one or more of the distances,which may correspond to a lower surface and an upper surface of the baseof the container, can be used to select an appropriate focal plane forimaging of samples in the sample container.

An example of a chromatic confocal device which could be used toimplement the displacement sensor is the optoNCDT 2400 Confocalchromatic displacement sensor manufactured by Micro-Epsilon ofKoenigbacher Str. 15 94496 Ortenburg, Germany. This device focusespolychromatic white light along an optical axis using a series of lenseswhich disperse the polychromatic white light into monochromatic light ata given point on the optical axis using the chromatic deviation of thelenses. Each wavelength has a particular distance assigned to it byfactory calibration. The light reflected back from a surfaceintersecting the optical axis is provided to a receiver via a confocalaperture which substantially restricts the light received by thereceiver to the light which was precisely focused at the intersectingsurface. The receiver then determines the intensity and wavelength ofthe received light and uses this information to determine the distancefrom the displacement sensor of an object intersecting its optical axis.

Referring to FIG. 8, a displacement sensor 410, which operates inaccordance with the arrangement shown in FIG. 7, is shown in use whendetermining a suitable focal length for imaging samples in a biologicalsample container 420. FIG. 8 also shows a camera 405 which is mountedtogether with the displacement sensor 410 on a mount 415. The camera 405is relatively positioned with the displacement sensor 410 to provide aone well offset, although it will be appreciated that alternativeoffsets could be used. In this way, the camera 405 is able to image onewell while the displacement sensor 410 detects the focal plane of thenext well. As with FIG. 7, the displacement sensor 410 of FIG. 8generates a continuum of focused points of monochromatic light ofdifferent wavelengths along an optical axis, of which five wavelengthsare illustrated, these being wavelength λ₁, wavelength λ₂, wavelengthλ₃, wavelength λ₄ and wavelength λ₅. The biological sample container 420in this case is a well plate which has a plurality of wells for holdingsamples. Each well has a wall 435 and a base 440. The wells each containa solution 437 containing samples 436 to be imaged by the camera 405.The top surface 438 of the solution 437 is also shown, and willgenerally have a meniscus region adjacent to the side wall, asschematically illustrated. The well to which the displacement sensor 410is currently being applied has a base portion with a lower surface 430and an upper surface 440. The base portion has a thickness d_(t) asmeasured between the lower surface 430 and the upper surface 440. It canbe seen that the focal point of wavelength λ₂ is intersected by thelower surface 430 of the base of the well, and that the focal point ofwavelength λ₃ is intersected by the upper surface 440 of the base of thewell. The focal points of wavelengths λ₁ and λ₄ are not intersected inthis case by any part of the biological sample container 420. However,the focal point of wavelength λ₅ intersects with the top surface of theliquid solution 438.

FIG. 8 also shows a schematic example graph of detected light intensityas a function of wavelength, which represents the spectrometer output ofthe displacement sensor 410 when the biological sample container 420 isat the position shown intersecting the optical axis of the focusedlight. The horizontal axis of the graph is marked with wavelengths λ₁,λ₂, λ₃, λ₄ and λ₅ which correspond to the like-referenced wavelengths ofthe focal points on the optical axis intersecting the biological samplecontainer. It can be seen from the graph that two intensity peaks arisewhich exceed a threshold intensity value I_(t), and that thesecorrespond to wavelengths λ₂ and λ₃. These two peaks therefore representthe position of the upper and lower surfaces of the base of thebiological sample container 420. All other wavelengths, including λ₁, λ4and λ₅, represented on the graph exhibit a level of intensity below thethreshold value I_(t) and are therefore ignored. However, the wavelengthλ₅ exhibits a lower intensity, broader peak due to its interception ofthe solution top surface 438. By virtue of the known relationshipbetween wavelength and position, the position of the upper and/or lowersurfaces of the base of the biological sample container 420 can bedetermined, and used to set a focal plane for imaging samples in thebiological sample container.

The most straightforward method of setting the focal plane is todirectly detect the upper surface of the base and use this to set thefocal plane, either by setting to focal plane at the upper surface ofthe base, or by adding a small offset to the upper surface of the baseto set the focal plane just above the upper surface within the containeritself. This method is most suitable where the position of the uppersurface can be determined reliably.

An alternative method of setting the focal plane is available if theupper surface of the base cannot be reliably detected. This may arisewhen the container is filled with water or a solution, or if some debrisexists on the upper surface of the base of the container. In this case,the signal from the upper surface may be either very weak due to therelatively small difference in refractive index between the plastic ofthe container to the solution held within the container, or unreliable.In order to overcome this problem, the lower surface of the base of thecontainer can be detected, and a thickness value identifying thethickness of the base of the container can be added to the lower surfaceposition to determine the position of the upper surface, and thus therequire focal plane position. This method is suitable where thevariation in thickness of the base of the container is small, and wherethe thickness information is available. The lower surface of the basecan be detected more easily in this case because of the largerdifference in refractive index, n, between the well plate material (e.g.n˜1.5-1.6) and air (n=1) compared with the refractive index differencebetween the well plate material and the liquid contained in the wells(e.g. aqueous solution) which will typically have a refractive indexclose to that of water (n˜1.33).

Known materials for well plates and other biological sample containersinclude various glasses, such as Pyrex (TM), and plastics compounds suchas polystyrene (PS), polypropylene (PP), polyethylene (PE), cycloolefin(co-) polymer (COP), styrene-acrylonitrile copolymer (SAN), polyamide(nylon), polyimide (PI), polycarbonate (PC), and polymethyl methacrylate(PMMA).

If the thickness of the base is not known in advance from manufacturersdata, it can be derived by detecting both the top and bottom surfaces ofthe base at a plurality of regions of the sample container, for instanceas a scan of the imaging area. The thickness data can be statisticallyprocessed to remove data having a large deviation from the mean, andthen averaged to obtain a mean value of thickness. This mean value ofthickness can be added to the position of the lower surface of the baseto identify the position of the upper surface of the base.

FIG. 9 schematically illustrates an example scanning pattern which canbe used by both the displacement sensor 410 and the imaging camera 405when a sequential work flow is used. In the sequential work flow, thedisplacement sensor scans across all wells in a well plate to identify asuitable focal plane position for each well, with the focal planeinformation being used to generate a focal plane profile. Then, when afocal plane has been determined for each well, the imaging camera scansacross all wells in the well plate, imaging each one in accordance withthe respective focal plane position identified in the focal planeprofile by suitable adjustment of the camera position with respect ofthe well. A simple array of twelve wells of a well plate is shown, in 3rows R1, R2 and R3 and 4 columns C1, C2, C3 and C4. The route of therelative movement between the well plate and the displacement sensor isshown. The scan, both for the displacement sensor and the imagingcamera, follows lines in the direction of the arrows. Thus, startingwith the upper left well, the wells are focused, and then imaged in thefollowing order: R1C1, R1C2, R1C3, R1C4, R2C4, R2C3, R2C2, R2C1, R3C1,R3C2, R3C3, R3C4 (row wise scanning).

FIG. 10 schematically illustrates an example scanning pattern which canbe used when an on-the-fly work flow is used. In the on-the-fly method,the displacement sensor and the imaging camera are offset from eachother which allows the displacement sensor to be scanned across theareas for imaging slightly ahead of the imaging camera, enabling a focalplane to be selected for imaging based on the detected base position andthen used to set the focus of the imaging camera as the camera isbrought into the position previously occupied by the displacementsensor.

As with FIG. 9, a simple array of twelve wells of a well plate is shown,in 3 rows R1, R2 and R3 and 4 columns C1, C2, C3 and C4. The route ofthe relative movement between the well plate and the displacement sensoris shown. The position of the displacement sensor follows lines in thedirection of the arrows. Thus, starting with the upper left well R1C1,the displacement sensor determines a focal plane for the well, and thenthe displacement sensor shifts slightly to the right as indicated by theshort arrow to enable the well to be imaged by the offset imagingcamera. The displacement sensor then moves to the second well R1C2,where the same process is repeated. In this case, the focusing andimaging order is: R1C1, R1C2, R1C3, R1C4, R2C1, R2C2, R2C3, R2C4, R3C1,R3C2, R3C3, R3C4 (raster scanning with flyback).

FIG. 11 schematically illustrates the sequential work flow method in anembodiment where the top surface is detected and used to set the focalplane of a well in a well plate. The process starts at a step S1 inwhich the displacement sensor is aligned with a well of the well plate.The displacement sensor is then operated at a step S2 to detect anddetermine the position of the top surface of the base of the well. At astep S3, the determine top surface position is used to set a focal planeposition for imaging of the well, and at a step S4 this focal planeposition is added to a focal plane profile in association with the welllocation. At a step S5 it is determined whether the focusing process hasfinished or whether there are further wells to be focused. If there arefurther wells to be focused then the process returns to the step S1where the displacement sensor is brought into alignment with anotherwell of the well plate. If there are no further wells to focus, then thefocusing process ends and the imaging process can commence. Inparticular, at a step S6 the imaging camera is aligned with the firstwell identified in the focal length profile, and at a step S7 theimaging camera is focused to the focal plane identified in thecorresponding entry in the focal plane profile, and then at a step S8 asample contained in the well is imaged. At a step S9 it is determinedwhether there are any more wells to image, or whether the imagingprocess has completed. If there are further wells to image, processingreturns to the step S6 where the imaging camera is aligned with anotherwell of the well plate. If there are no further wells to image, thenprocessing terminates.

FIG. 12 schematically illustrates the sequential work flow method in anembodiment where the bottom surface is detected and used in combinationwith derived thickness information to set the focal plane of a well in awell plate. The process can be considered in three stages, the firstbeing a thickness measurement stage, the second being a focusing stage,and the third being an imaging stage. The process starts with athickness measurement stage and a step U1 in which the displacementsensor is aligned to a well in the well plate. At a step U2 both theupper and lower surfaces of the base of the well are detected and theirlocations are determined. At a step U3 the separation between thedetected upper surface position and lower surface position, whichrepresents the thickness of the base at that point, is determined. At astep U4 it is determined whether any further thickness measurements areto be made, and if so the process returns to the step U1 where thedisplacement sensor is aligned to another region of the well plate,either at a different point in the same well, or at a different well. Ifit is determined at the step U4 that no further thickness measurementsare to be made, the process moves on to a step U5, where the thicknessmeasurements are filtered to remove measurements which deviate togreatly from the mean thickness measured. This can be achieved bycomparing the deviation of each thickness measurement from the mean witha predetermined deviation threshold. Then, the filtered data is averagedat a step U6 to determine an average thickness for the well plate base.

Then, processing moves on to the focusing stage, where the displacementsensor is aligned with a well of the well plate at a step U7. Thedisplacement sensor is then used to determine the position of the bottomsurface of the base at a step U8, and the position of the bottom surfaceis then used to set the position of the focal plane at a step U9 byadding the derived thickness information to the bottom surface position.At a step U10, the focal plane position is added to the focal planeprofile. At a step U11 it is determined whether the focusing process hasfinished or whether there are further wells to be focused. If there arefurther wells to be focused then the process returns to the step U7where the displacement sensor is brought into alignment with anotherwell of the well plate. If there are no further wells to focus, then thefocusing stage ends and the imaging stage can commence. The imagingstage described by steps U12 to U16 corresponds exactly to the steps S6to S10 of FIG. 11, and will thus not be described further.

FIG. 13 schematically illustrates the on-the-fly work flow method in anembodiment where the top surface is detected and used to set the focalplane of a well in a well plate. At a step V1 the displacement sensor isaligned with a well of the well plate. The displacement sensor detectsthe top surface of the base of the well at a step V2 and determines itsposition. The position of the top surface is used at a step V3 to set afocal plane for the well to be imaged. Then, at a step V4 an imagingcamera offset from the displacement sensor is aligned with the wellwhile the imaging camera is focused to the determined focal planeposition at a step V5. At a step V6 a sample in the well is imaged atthe selected focal plane position. At a step V7 it is determined whetherthere are any further wells to image, or whether the process hasfinished. If there are further wells to image, the process returns tothe step V1, where the displacement sensor is aligned with a furtherwell. If there are no further wells to image, processing ends.

FIG. 14 schematically illustrates the on-the-fly work flow method in anembodiment where the bottom surface is detected and used in combinationwith known thickness information to set the focal plane of a well in awell plate. At a step W1 a displacement sensor is aligned by a well ofthe well plate. At a step W2 the bottom surface of the base is detectedby the displacement sensor and its position determined. The position ofthe bottom surface is used at a step W3 in combination with knownthickness information, for instance provided by the well platemanufacturer, to set a focal plane for the well to be imaged. This canbe achieved by adding the thickness of the plate to the determinedposition of the bottom surface of the well to determine the top surfaceof the well and thus a suitable focal plane for imaging. Then, at a stepW4 an imaging camera offset from the displacement sensor is aligned withthe well while the imaging camera is focused to the determined focalplane position at a step W5. At a step W6 a sample in the well is imagedat the selected focal plane position. At a step W7 it is determinedwhether there are any further wells to image, or whether the process hasfinished. If there are further wells to image, the process returns tothe step W1, where the displacement sensor is aligned with a furtherwell. If there are no further wells to image, processing ends.

For each embodiment of the imaging and scanning described above, theapparatus is preferably controlled by a controller such as a computer,to provide automated handling of biological sample containers.

In the above description, the optical source and detecting camera aremainly described as an illumination and imaging system without referenceto spectroscopic properties. However, it will be understood thatspectroscopic aspects can be incorporated into the apparatuses andmethods of the invention. For example, the imaging may be offluorescence or Raman properties.

Embodiments of the present invention may work particularly well ifcombined with the vacuum suction method described with reference toFIGS. 4A and 4B and in US20070009395A1 [3]. In particular, by flatteningthe base of the biological sample container against an optical flat, itis possible to use the chromatic focal plane sensor of the presentinvention to measure the thickness of the base of the container inconjunction with the position of the base as defined by the optical flaton which it is held down by vacuum suction. US20070009395A1 [3] is thusincorporated herein by reference in its entirety, in particular itsdetailed description, associated figures, claims and abstract.

It will be appreciated that although particular embodiments of theinvention have been described, many modifications/additions and/orsubstitutions may be made within the spirit and scope of the presentinvention.

REFERENCES

-   [1] U.S. Pat. No. 6,130,745 (Manian et al)-   [2] U.S. Pat. No. 6,441,894 (Manian et al)-   [3] US20070009395A1 (Genetix Limited)-   [4] Micro-Epsilon product guide for the OptoNCDT 2400 Confocal    Chromatic Displacement Sensor (Micro-Epsilon of Koenigbacher Str. 15    94496 Ortenburg, Germany)-   [5] US2006164644A1 (Genetix Limited)-   [6] US2006166305A1 (Genetix Limited)

1. An apparatus for optically analyzing samples in a biological samplecontainer, the biological sample container comprising a plurality ofsamples located at respective sample locations distributed over thebiological sample container, each sample location being coincident with,or vertically offset from, a base of the biological sample container,wherein the base is defined by upper and lower surfaces, the apparatuscomprising: a container station in which a biological sample containercan be arranged; an optical acquisition device comprising a detector andan objective arranged to view a biological sample container arranged inthe container station from below, the positions of the detector andobjective collectively defining a focal plane for optical acquisition; afocal plane sensor arranged to view a biological sample containerarranged in the container station from below and comprising: (i) apolychromatic light source operable to generate polychromatic light overa range of wavelengths; (ii) a focusing arrangement with defined axialchromatism arranged to focus respective wavelengths of the polychromaticlight to respective pre-calibrated positions along an axis that extendsthrough where the base of a biological sample container arranged in thecontainer station would be; (iii) a detection unit comprising aspectrometer arranged to spectrally isolate components of saidpolychromatic light scattered from said optical axis; and (iv) a dataprocessing unit operable to determine the position of at least one ofthe upper and lower surface of the base from the isolated components ofthe scattered polychromatic light output by the spectrometer; apositioning apparatus operable to adjust the focal plane of the opticalacquisition device relative to the container station; and a controlleroperable to control the focal plane sensor, optical acquisition deviceand positioning apparatus to: (i) determine a desired focal plane foreach sample location with reference to the determined position of the atleast one of the upper and lower surface of the base at that samplelocation; and (ii) use the optical acquisition device to collect lightfrom each of the sample locations with the focal plane adjusted to itsdesired setting.
 2. The apparatus of claim 1, wherein the detection unitfurther comprises a confocal collection aperture arranged to pass lightscattered from said optical axis to the spectrometer.
 3. The apparatusof claim 1, wherein the controller is operable to determine a desiredfocal plane for each of a plurality of the sample locations prior tocollecting light using the optical acquisition device from those samplelocations.
 4. The apparatus of claim 1, wherein the controller isoperable to determine a desired focal plane for a sample location andcollect light from that location using the optical acquisition deviceprior to moving to the next sample location.
 5. The apparatus of claim1, wherein the controller is operable to take the focal plane for eachsample location with reference to the measured upper surface position,if available, and otherwise with reference to the measured lower surfaceposition.
 6. The apparatus of claim 1, wherein the controller isoperable to take the focal plane for each sample location with referenceto a support surface on which the base is in contact offset by a basethickness value computed from the difference between the measured upperand lower surface positions, if available, and otherwise with referenceto the support surface.
 7. The apparatus of claim 1, wherein thedetector is an array detector for imaging the sample location.
 8. Theapparatus of claim 1, wherein the sample locations are defined bypositions of wells in a standard well plate.
 9. The apparatus of claim1, wherein the container station is adapted to accommodate standard wellplates having standard external dimensions.
 10. The apparatus of claim1, wherein the controller is operable to control the focal plane sensor,optical acquisition device and positioning apparatus assuming that thebiological sample container is a well plate having a standard number ofwells distributed in standard positions over the well plate.
 11. Theapparatus of claim 1, wherein the controller is operable to control thefocal plane sensor, optical acquisition device and positioning apparatusassuming that the sample locations are positions of single cells or cellcolonies provided by a cell or cell colony imager.
 12. A method ofoptically analyzing samples in a biological sample container, comprisingthe steps of: providing a biological sample container containing aplurality of samples located at respective sample locations distributedover the biological sample container, each sample location beingcoincident with, or vertically offset from, a base of the biologicalsample container, wherein the base is defined by upper and lowersurfaces; providing an optical acquisition device comprising a detectorand an objective which collectively define a focal plane for opticalacquisition; measuring the position of at least one of the upper andlower surface of the base at each of the sample locations by focusing acontinuum of wavelengths of polychromatic light to a continuum ofrespective pre-calibrated positions along an axis extending through thebase, and by collecting and spectrally decomposing those components ofthe polychromatic light scattered from said axis; and collecting lightfrom each of the sample locations by adjusting the focal plane to becoincident with, or vertically offset from, the upper surface of thebase based on the position of the at least one of the upper and lowersurface of the base measured at that sample location.
 13. The method ofclaim 12, wherein the components of the polychromatic light scatteredfrom said axis are collected through a confocal aperture to isolate thepolychromatic light scattered from said axis.
 14. The method of claim12, wherein the measuring step is carried out for a plurality of thesample locations prior to carrying out the collecting step on thosesample locations.
 15. The method of claim 12, wherein the measuring andcollecting steps are carried out at each sample location prior to movingto the next sample location.
 16. The method of claim 12, wherein thefocal plane for each sample location is taken with reference to themeasured upper surface position, if available, and otherwise withreference to the measured lower surface position.
 17. The method ofclaim 12, wherein the focal plane for each sample location is taken withreference to a support surface on which the base is in contact offset bya base thickness value computed from the difference between the measuredupper and lower surface positions, if available, and otherwise withreference to the support surface.
 18. The method of claim 12, whereinthe detector is an array detector for imaging the sample location. 19.The method of claim 12, wherein the sample locations are wells of a wellplate.
 20. The method of claim 12, wherein the sample locations arepositions of individual cells or cell colonies.
 21. The method of claim12, wherein light is collected from each of the sample locations bymoving the sample container from sample location to sample location. 22.The method of claim 12, wherein light is collected from each of thesample locations by moving the optical acquisition device from samplelocation to sample location.
 23. The method of claim 12, wherein theoptical acquisition device further comprises a light source.
 24. Themethod of claim 23, wherein the light source is switched on and off toprovide illumination for each image at each of the sample locations. 25.The method of claim 23, wherein the optical acquisition device furthercomprises a camera with a shutter, and wherein light is collected fromeach of the sample locations by opening the camera shutter to determinethe exposure.
 26. The apparatus of claim 1, wherein the controller isoperable to control the optical acquisition device and positioningapparatus to collect light from each of the sample locations by movingthe sample container from sample location to sample location.
 27. Theapparatus of claim 1, wherein the controller is operable to control theoptical acquisition device and positioning apparatus to collect lightfrom each of the sample locations by moving the optical acquisitiondevice from sample location to sample location.
 28. The apparatus ofclaim 1, wherein the optical acquisition device further comprises alight source.
 29. The apparatus of claim 28, wherein the controller isoperable to switch the light source on and off to provide illuminationfor each image at each of the sample locations.
 30. The apparatus ofclaim 28, wherein the optical acquisition device further comprises acamera with a shutter, and wherein the controller is operable to controlthe optical acquisition device to collect light from each of the samplelocations by opening the camera shutter to determine the exposure.